Huang et al. BMC Biology (2019) 19:419 https://doi.org/10.1186/s12870-019-1997-2

RESEARCH ARTICLE Open Access Transcriptomic analysis of vesicaria subs. sativa lines with contrasting tolerance to polyethylene glycol-simulated drought stress Bang-Lian Huang1,2, Xuan Li1, Pei Liu1, Lan Ma1, Wenhua Wu1, Xuekun Zhang3, Zaiyun Li4 and Bangquan Huang1*

Abstract Background: Eruca vesicaria subsp. sativa is one of the Cruciferae species most tolerant to drought stress. In our previous study some extremely drought-tolerant/sensitive Eruca lines were obtained. However little is known about the mechanism for drought tolerance in Eruca. Methods: In this study two E. vesicaria subs. sativa lines with contrasting drought tolerance were treated with liquid MS/PEG solution. Total RNA was isolated from 7-day old whole seedlings and then applied to Illumina sequencing platform for high-throughput transcriptional sequencing. Results: KEGG pathway analysis indicated that differentially expressed genes (DEGs) involved in alpha-Linolenic acid metabolism, Tyrosine metabolism, Phenylalanine, Tyrosine and tryptophan biosynthesis, Galactose metabolism, Isoquinoline alkaloid biosynthesis, Tropane, Piperidine and pyridine alkaloid biosynthesis, absorption, were all up-regulated specifically in drought-tolerant (DT) Eruca line under drought stress, while DEGs involved in ribosome, ribosome biogenesis, Pyrimidine metabolism, RNA degradation, Glyoxylate and dicarboxylate metabolism, Aminoacyl-tRNA biosynthesis, Citrate cycle, Methane metabolism, Carbon fixation in photosynthetic organisms, were all down-regulated. 51 DEGs were found to be most significantly up-regulated (log2 ratio ≥ 8) specifically in the DT line under PEG treatment, including those for ethylene-responsive transcription factors, WRKY and bHLH transcription factors, calmodulin-binding transcription activator, cysteine-rich receptor-like protein kinase, mitogen- activated protein kinase kinase, WD repeat-containing protein, OPDA reductase, allene oxide cyclase, aquaporin, O- acyltransferase WSD1, C-5 sterol desaturase, transporter ERD6-like 12, trehalose-phosphate phosphatase and galactinol synthase 4. Eight of these 51 DEGs wre enriched in 8 COG and 17 KEGG pathways. Conclusions: DEGs that were found to be most significantly up-regulated specifically in the DT line under PEG treatment, up-regulation of DEGs involved in Arginine and proline metabolism, alpha-linolenic acid metabolism and down-regulation of carbon fixation and protein synthesis might be critical for the drought tolerance in Eruca. These results will be valuable for revealing mechanism of drought tolerance in Eruca and also for genetic engineering to improve drought tolerance in crops. Keywords: Eruca vesicaria subs. Sativa, Genotypes, Drought tolerance, RNA-seq, Gene expression

* Correspondence: [email protected] 1Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, College of Life Science, Hubei University, Wuhan 430062, China Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Plant Biology (2019) 19:419 Page 2 of 11

Background Table 1 Illumina RNA-Seq reads and de novo assembly statistics Drought is one of the most important environmental of Eruca transcriptome stresses in the world [1–3]. Previous studies indicated that Total number of raw reads 314,322,210 plant responses to drought stress is quite complicated and Total number of clean reads 254,660,002 exploring drought tolerance mechanism in is still a Mean length of reads (bp) 90 challenge [4–7]. Next generation sequencing (NGS) using Number of total unigenes 430,166 Illumina HiSeq2000 covers the transcriptome many folds and allows quantification of the detected transcripts [8, 9]. Mean length of unigenes (bp) 511.56 By using next generation sequencing the gene expression is Minimum unigene length (bp) 201 quantified, making it possible for quantitative comparisons Maximum unigene length (bp) 16,517 [10]. Many researchers have used transcriptome sequencing N50 (bp) 638 – to study the responses to drought stress in crops [10 16]. Unigenes annotated to Nr 139,090 Biological and genetic diversity exists among and within Unigenes annotated to KEGG 40,463 plant species regarding drought tolerance, however many of these adaptive mechanisms are not completely under- Unigenes annotated to COG 105,052 stood. Genome-wide identification of drought-responsive − transcriptions using genotypes with contrasting drought- value < 10 5)(Fig.1). Among the unigenes 83,392 had tolerance will help revealing interactions among metabolic identity between 22.86–80% and 55,698 higher than 80%. pathways in response to drought stress [12]. Eruca vesi- Regarding the species distribution, 83,034 unigene caria subsp. sativa is one of the Cruciferae species most (59.70%) had similarity between 19.32–80%, 56, 056 uni- tolerant to drought stress [17, 18]. In our previous study gene (40.30%) had similarity higher than 80%. Of the 139, some extremely drought-tolerant/sensitive Eruca lines were 090 unigenes annotated to Nr, 36,254 (26.06%) had obtained [19]. In this study, Illumina sequencing technology matches to sequences from Brassica napus, followed by was used to identify the differentially expressed genes that from Brassica rapa (27,894, 20.05%), Eutrema salsugi- (DEGs) and their biochemical pathways related to drought neum (5366, 3.86%), Hordeum vulgare (3578, 2.57%), Ara- tolerance in drought tolerant (DT) versus drought- bidopsis thaliana (3391, 2.44%), Physcomitrella patens susceptible (DS) Eruca genotypes. Expression level of some (3289, 2.36%), Camelina sativa (3249, 2.34%) (Fig. 2). selected drought-responsive genes was validated by using quantitative RT-PCR. To our knowledge,thisisthefirstre- COG annotation and enrichment port about the whole transcriptome analysis in E. vesicaria In total 105,052 unigenes were anotated to COG (Clusters subsp. sativa. The results provide transcriptome data valu- of Orthologous Groups of proteins) database and assigned able for understanding drought tolerance mechanism in to 25 COG functional clusters (Fig. 3), among which the Eruca and the differentially expressed genes could be valu- “general function prediction only” cluster comprised the able for improving drought tolerance in crops. highest number of unigenes (15,472, 14.73%), followed by the cluster “translation, ribosomal structure and biogen- Results esis” (14,062, 13.39%) and “Posttranslational modification, Overview of Illumina RNA sequencing and de novo protein turnover, chaperones” (10,244, 9.75%). By contrast, assembly of E. vesicaria subs. sativa transcriptome only 110 unigenes were classified into “Cell motility”,98 In total 314,322,210 raw reads after sequencing produced unigenes classified into “Nuclear structure” and 3 uni- 254,660,002 clean reads including 430,166 unique genes with genes classified into “Extracellular structures” (Fig. 3). N50 value of 638 bp and average length of 511.56 bp. The unigene length ranged from 201 to 16, 517 bp (Table 1). The unigenes included 94,463 clusters and 335,703 sin- GO anotation and enrichment gletons. Among the unigenes there were 308,310 (71.67%) GO (Gene ontology) with Nr annotation indicated that with length from 200 to 500 bp, 76,255 (17.73%) with 4953 unigenes were classified into 69 groups that could length from 500 bp to 1000 bp, 24,662 (5.73%) with length be categorized into three main classifications: “biological from 1000 bp to 1500 bp, 11,229 (2.61%) with length from process” (2859), “cellular component” (643) and “mo- 1500 bp to 2000 bp, 9709 (2.26%) with length longer than lecular function” (1451). In the “molecular function” 2000 bp, and no unigenes shorter than 200 bp (Table 2). class the largest number of unigenes (984) were involved Based on Nr annotation and the E-value distribution, it in “catalytic activity”; in the “cellular component” class was found that among the annotated unigenes 51,486 the largest number of unigenes (168) were involved in − (37.02%) shared very strong homology (E-value < 10 60), “cell part”; in the “biological process” class the largest − 31,700 (22.80%) had strong homology (10 60

Table 2 Length distribution of the Unigenes Length (nt) Total < 200 200–500 500–1000 1000–1500 1500–2000 ≥2000 Number of Unigenes 430,166 0 308,310 76,255 24,662 11,229 9709 % 100 0.00 71.67 17.73 5.73 2.61 2.26

KEGG pathway annotation and unigene enrichment metabolism, 39 in Plant hormone signal transduction, 21 In total 28,295 unigenes were annotated to 344 KEGG path- in Phenylpropanoid biosynthesis, 13 in alpha-Linolenic ways (Additional file 2). The number of unigenes in different acid metabolism and 41 in D-Glutaminehe D-glutamate KEGG pathways ranged from 1 to 3245. The map with the metabolism (Additional file 4). highest unigene representation was Ribosome (ko03010, Common pathways betweem DS-MS vs DS-PEG and 3245 unigenes, 11.47%), followed by Carbon metabolism DT-MS vs DT-PEG are Plant-pathogen interaction, (ko01200, 1483 unigenes, 5.24%), Biosynthesis of amino Phenylalanine metabolism, Plant hormone signal acids (ko01230, 1360 unigenes, 4.81%), Protein processing in transduction, Ribosome, Phenylpropanoid biosynthesis, endoplasmic reticulum (ko04141, 1230 unigenes, 4.35%). D-Glutamine and D-glutamate metabolism; Pathways specifically in DS-MS vs DS-PEG are Drug metabolism- KEGG pathways and DEGs related to drought tolerance cytochrome P450, Atrazine degradation, Tyrosine me- Difference in gene expression between drought-tolerant/ tabolism, Metabolism of xenobiotics by cytochrome sensitive Eruca lines under MS/PEG treatment was ana- P450, Biosynthesis of siderophore group nonribosomal pep- lyzed by mapping back to the previous de novo assembling tides, Chemical carcinogenesis, Glutathione metabolismand results using RSEM. As shown in Fig. 4 there were 5560, Lysosome; pathways specifically in DT-MS vs DT-PEG are 4654, 9605, 8979 and 13,951 unigenes showing differential Arginine and proline metabolism and alpha-Linolenic acid expression including both up- and down-regulated uni- metabolism (Additional files 3 and 4). genesinDTvsDS,DT-MSvsDT-PEG,DS-MSvsDS- Referring to Audic’salgorithm[20], FDR (False Discov- PEG, DS-MS vs DT-MS, DS-PEG vs DT-PEG, respectively. ery Rate) was calculated based on the P-value that corre- In the DS-MS vs DS-PEG group, 646 DEGs were sponds to differential expression tests of transcripts. By enriched in 14 KEGG pathways (Q value < 0.05), i.e., 405 in using “FDR ≤ 0.05 and the absolute value of log2 Ratio ≥1” Ribosome, 32 in Drug metabolism-cytochrome P450, 32 in as the threshold, we identified in the DS-MS vs DS-PEG Plant-pathogen interaction, 26 in Tyrosine metabolism, 40 group 3287 unigenes up-regulated and 6318 down- in Phenylalanine metabolism, 37 in Plant hormone signal regulated; in the DT-MS vs DT-PEG couple, 2299 uni- transduction, 32 in Metabolism of xenobiotics by cyto- genes up-regulated and 2355 down-regulated (Fig. 4). It chrome P450, 30 in Chemical carcinogenesis, 36 in Phenyl- was specifically in DT-MS vs DT-PEG but not in DS-MS propanoid biosynthesis, 47 in Glutathione metabolism, 54 vs DS-PEG that 2946 DEGs were detected, among which in Lysosome, 7 in D-Glutamine and D-glutamate metabol- 1584 were up-regulated and 1362 down-regulated, 449 ism, 3 in Biosynthesis of siderophore group nonribosomal had Nr annotation and 552 annotated to 232 KEGG path- peptides and 1 in Atrazine degradation (Additional file 3). ways (data not shown). In the DT-MS vs DT-PEG group, 395 DEGs were KEGG pathway analysis indicated that in DT-MS vs enriched in 8 KEGG pathways (Q value < 0.05), i.e., 257 DT-PEG but not in DS-MS vs DS-PEG, DEGs involved in in ribosome, 42 in Plant-pathogen interaction, 20 in alpha-Linolenic acid metabolism, Tyrosine metabolism, Phenylalanine metabolism, 31 in Arginine and proline Phenylalanine, tyrosine and tryptophan biosynthesis,

Fig. 1 Homology search of unigenes against the Nr database Fig. 2 Species distribution of Unigenes in the Nr database Huang et al. BMC Plant Biology (2019) 19:419 Page 4 of 11

Fig. 3 COG Functional classification and distribution

Galactose metabolism, Isoquinoline alkaloid biosynthesis, cycle, Methane metabolism, Carbon fixation in photosyn- Tropane, piperidine and pyridine alkaloid biosynthesis, thetic organisms, were all down-regulated (log2 Ratio ≤−1). Mineral absorption, were all up-regulated (log2 Ratio ≥ 1); Most DEGs involved in Porphyrin and chlorophyll metabol- DEGs involved in ribosome, ribosome biogenesis, Pyrimi- ism, Ubiquinone and other terpenoid-quinone biosynthesis, dine metabolism, RNA degradation, Glyoxylate and dicar- Arachidonic acid metabolism, Glutathione metabolism, gly- boxylate metabolism, Aminoacyl-tRNA biosynthesis, Citrate cerophospholipid metabolism, Phenylalanine metabolism,

Fig. 4 Significantly and differentially expressed genes between drught-tolerant/ sensitive Eruca with PEG/MS treatment Huang et al. BMC Plant Biology (2019) 19:419 Page 5 of 11

Plant-pathogen interaction, were up-regulated (log2 Ratio ≥ genes. Identification of DEGs exclusively in the tolerant 1); Most DEGs involved in Cell cycle, Peroxisome, Carbon genotype would be valuable for revealing the mechanisms metabolism, protein processing in ER, Regulation of actin responsible for stress tolerance [21]. In the present study cytoskeleton, Arginine and proline metabolism, Pyruvate we provided detailed transcriptomic profiles of whole seed- metabolism, Pentose phosphate pathway, Nitrogen metabol- lings of two Eruca lines with contrasting drought tolerance. ism, Spliceosome, Apoptosis, Lysine degradation, lysosome, signaling pathway, MAPK signaling pathway, Pro- ABA responses and stress signaling panoate metabolism, Valine, leucine and isoleucine degrad- Drought stress induces ABA accumulation, which in ation, Alanine, aspartate and glutamate metabolism, turn leads to stomatal closure to keep the water status in Proteasome, Purine metabolism, RNA transport, Oxidative plants [22–24]. Evidence indicates that WRKY proteins, phosphorylation, down-regulated (Additional file 5). including those induced by ABA, are upregulated under Among the 2946 DEGs detected specifically in DT-MS vs drought stress in rice [25]. OsWRKY45 overexpression DT-PEG but not in DS-MS vs DS-PEG, 1890 had blast hits increased drought tolerance [26], while activated expres- against the NCBI database (Additional file 6), among which sion of WRKY57 resulted in drought tolerance in Arabi- 51 DEGs were most significantly up-regulated (log2 Ratio ≥ dopsis [27]. In this study 19 of 26 DEGs for WRKY 8), including 3 DEGs for trehalose-phosphate phosphatase, transcription factor were significantly up-regulated spe- 2 for allene oxide cyclase, 2 for ethylene-responsive tran- cifically in the drought tolerant Eruca line under scription factor, 1 for aquaporin TIP1–1, 2 for sugar trans- drought stress (hereafter referred as significantly up- porter ERD 6-like 12, 1 for transcription factor MYC3-like, regulated), among which one for WRKY transcription 1 for senescence-associated carboxylesterase 101-like, 1 for factor 55-like (c195662 g1 i2) was most significantly up- calmodulin-binding transcription activator 2, 1 for cysteine- regulated specifically in drought-tolerant Eruca line PI rich receptor-like protein kinase 17, 1 for galactinol syn- 426649 under PEG-simulated drought stress (hereafter thase 4, 1 for mitogen-activated protein kinase kinase 4, 1 referred as most significantly up-regulated). for transcription factor bHLH19-like, 1 for C5-sterol desa- Basic helix-loop-helix (bHLH) genes are important in phy- turase, 1 for WD repeat-containing protein, 1 for WRKY tohormone signaling. Rice OsbHLH148 confers drought tol- transcription factor, 1 for O-acyltransferase WSD1, 2 for erance by interacting with OsJAZ proteins [28]. Arabidopsis low-temperature-induced 65 kDa protein; 275 unigenes bHLH122 and PebHLH35 from Populus euphratica are were most significantly down-regulated (log2 Ratio ≤− positive regulators of drought and salt tolerance and osmotic 8), including 38 DEGs for ribosomal protein. Fourteen signaling [29, 30]. In this study 5 DEGs for transcription fac- of the 51 most significantly up-regulated DEGs were tor bHLH were down-regulated and 8 up-regulated, among enriched in 8 COG pathways and 7 in 17 KEGG which one for transcription factor bHLH19-like (c196938 g3 pathways (Additional file 7 and 8). i6) was most significantly up-regulated (Additional file 6).

RNA-seq validation: qRT-PCR analysis Ethylene-responsive transcription factors Quantitative RT-PCR (qRT-PCR) assays were carried out Previous studies have indicated that Ethylene-responsive for 16 selected genes to verify the RNA-seq results. ERF4 transcription factors (ERFs) participate in abiotic stress (c189274 g1 i1), MLO2 (c205019 g2 i1), Ferritin (c194336 responses in plants [31]. Transcription of GhERF4 was g1 i1), receptor-like protein kinase (c203474 g1 i1), dehydrin increased rapidly Gossypium hirsutum when plants were ERD14 (c194017 g1 i1), disease resistance protein RPP-13 exposed to salt stress [32], and overexpression of ERF in- (c207395 g1 i3), O-acyltransferase WSD1-like (c175262_g2_ creased drought tolerance [33–36]. In this study 17 of i1), aquaporin TIP1 (c194457_g2_i2), pre-mRNA-splicing the 18 DEGs for ethylene-responsive transcription factor factor CLF1 (c161655_g2_i1), transporter were up-regulated, among which two for ERF056 (c197830_g3_i1), senescence-associated carboxylesterase (c163592 g1 i1, c163592 g2 i1) were most significantly (c191638_g3_i8), transcription factor MYC3 (c187590_g3_ up-regulated (Additional file 6). i3), EARLY FLOWERING 3 (c200550_g2_i1), were found to be up-regulated in DT-MS vs DT-PEG by qRT-PCR; proline Antioxidants and ROS modulation dehydrogenase (c201592 g1 i1); NRT1 (c193030 g1 i3), ribo- Reactive oxygen species (ROS) often acts as secondary some (c178914 g1 i1), were found to be down-regulated in messenger modulating stomatal closure in responses to DT-MS vs DT-PEG by qRT-PCR. The qRT-PCR results are different stimuli [22, 37]. In Arabidopsis, calmodulin- consistent with the RNA-seq results (Fig. 5 and 6). binding transcription activator 1 (AtCAMTA1) is in- volved in regulation of membrane integrity by inducing Discussion ABA responses to drought stress. The camta1 mutants Comparative study using lines with contrasting drought- are highly susceptible to drought stress [38]. In this tolerance is a useful tool for identifying drought-responsive study 10 DEGs for calmodulin-binding protein were all Huang et al. BMC Plant Biology (2019) 19:419 Page 6 of 11

Fig. 5 Quantitative RT-PCR confirmation of some up-regulated Unigenes Axis Y represents the relative quantification (RQ) between the PEG and MS treated seedlings, while the error bars indicate standard deviation of three replicates. 1a:c189274_g1_i1-PEG; 1b:c189274_g1_i1-MS; 2a:c205019_g2_i1-PEG; 2b:c205019_g2_i1- MS; 3a:c194336_g1_i1-PEG; 3b:c194336_g1_i1-MS; 4a:c203474_g1_i1-PEG; 4b:c203474_g1_i1-MS; 5a:c194017_g1_i1; 5b:c194017_g1_i1; 6a:c207395_g1_i3-PEG; 6b:c207395_g1_i3-MS; 7a:c175262_g2_i1-PEG; 7b:c175262_g2_i1-MS; 8a:c194457_g2_i2-PEG; 8b:c194457_g2_i2-MS; 9a:c161655_g2_i1-PEG; 9b:c161655_g2_i1-MS; 10a:c197830_g3_i1-PEG; 10b:c197830_g3_i1-MS; 11a:c191638_g3_i8-PEG; 11b:c191638_g3_i8-MS; 12a:c187590_g3_i3-PEG; 12b:c187590_g3_i3-MS; 13a:c200550_g2_i1-PEG; 13b:c200550_g2_i1-MS

up-regulated, among which one for calmodulin-binding It has been shown that MAPKs are involved in plant transcription activator 2-like (c195596 g1 i7) was most signal transduction in responses to different environ- significantly up-regulated (Additional file 6). mental stimuli [42–46]. MAPK, MAPKK and MAPKK Mos Cysteine-rich receptor-like kinases (CRKs) are kinase constitute a functional MAPK cascade. Activation regulated by ROS [39]. It was found that CRK45 posi- of MAPK helps its translocation to nucleus to phosphor- tively regulated plant responses to drought and salt ylate and activate transcription factors [47]. AtMKK1 in stresses by inducing expression of ABA-responsive and Arabidopsis activates AtMPK3 to transfer abiotic stess stress-inducible genes. CRK45 overexpression enhanced signals [48]. NtMEK2 (MAPKK) activates WIPK and drought tolerance in plants [40]. Overexpression of Ara- SIPK for drought signal transmission [49]. Overexpres- bidopsis CRK5 also increased ABA sensitivity and thus sion of GhMKK1 increased drought tolerance in Nicoti- enhanced drought tolerance [41]. In this study 10 of 12 ana benthamiana [41]. Overexpression of AtMKK1 in DEGs for cysteine-rich receptor-like protein kinase were Arabidopsis decreased ROS levels and increased drought up-regulated, among which one for cysteine-rich tolerance, while AtMKK1 deficiency resulted in elevated receptor-like protein kinase 17 (c196393 g1 i2) was most ROS and increased sensitivity to drought tolerance [50]. significantly up-regulated (Additional file 6). Plants over expressing AtMKK4 accumulated fewer ROS and showed less water-loss under drought stress [51]). In this study one DEG for mitogen-activated protein kinase kinase 4 (c198487 g1 i1) was most significantly up-regulated (Additional file 6). NAC genes such as SNAC3 contributes to drought re- sistance and osmotic modulation independent of ABA [52]. SNAC3 could interact with WD domain-containing protein to adjuste ROS in rice. In this study all 13 DEGs for NAC domain-containing proteinwereup-regulatedandone DEG for WD repeat-containing protein 53 (c198319 g1 i2) was most significantly up-regulated (Additional file 6).

Alpha-linolenic acid, jasmonate signaling and cell Fig. 6 Quantitative RT-PCR confirmation of some down-regulated Unigenes Axis Y represents the relative quantification (RQ) between membrane stability the PEG and MS treated Eruca seedlings, and the error bars indicate Plants keep membrane fluidity and integrity by modulating standard deviation of three replicates. 1a: proline dehydrogenase oleic and linolenic acid levels during stresses [53]. Alpha- (c201592 g1 i1)-PEG; 1b: proline dehydrogenase (c201592 g1 i1)-MS; linolenic acid is a major precursor for messengers including 2a: NRT1 (c193030 g1 i3)-PEG; 2b: NRT1 (c193030 g1 i3)- MS; 3a: jasmonic acid generated by oxidative modifications [54–56]. ribosome (c178914 g1 i1)-PEG; 3b: ribosome (c178914 g1 i1) – MS Antisense expression of Arabidopsis omega-3 fatty acid Huang et al. BMC Plant Biology (2019) 19:419 Page 7 of 11

desaturase gene led to reduced salt/drought tolerance in Trehalose is also important in protecting plants from tobacco [57], while over-expression of FAD3 or FAD8 re- stresses [77]. Trehalose-6-phosphate (T6P) is suggested to sulted in increased drought tolerance [58]. Lenka et al. [12] act as signaling metabolite responsing to the environment found that eight enzymes involved in alpha-linolenic acid [78]. In plants T6P is dephosphorylated by trehalose-6- metabolism were significantly induced by drought stress in phosphate phosphatase to produce trehalose [79]. In this drought-tolerant rice. In this study we also found all DEGs study all 6 DEGs for trehalose-phosphate phosphatase involved in alpha-linolenic acid metabolism up-regulated were up-regulated, among which 3 were most-up- (Additional file 5). regulated (Additional file 6). Jasmonic acid (JA) plays critical role in stomatal closure Galactinol synthase (GolS) plays important roles in car- during drought stress [59]. It is also involved in the produc- bon partitioning between sucrose and RFOs and the GolS tion of antioxidants regulating ascorbate and glutathione gene products are reported to function in responses to metabolism [60]. Based on the pathway established by Vick abiotic stresses [80–83]. Transgenic Arabidopsis and Bra- and Zimmerman [61], JA is produced from alpha-linolenic chypodium distachyon overexpressing AtGolS2 accumu- acid, and in this process allene oxide cyclase and OPDA re- lated more galactinol and raffinose and showed enhanced ductase (OPR) play important roles [62–66]. In this study drought tolerance [84, 85]. In this study one unigene for all four DEGs of OPDA reductase and 6 DEGs for allene galactinol synthase 4 (c194151 g2 i3) was most signifi- oxide cyclase were up-regulated, among which two DEGs cantly up-regulated (Additional file 6). for allene oxide cyclase (c184414 g1 i1, c184414 g2 i1) were most significantly up-regulated (Additional file 6). Down-regulation of carbon fixation and protein synthesis Previous studies indicated that Aquaporins (AQPs) Lenka et al. [12] found that under drought stress carbon played an important role in decreasing ion leakage (IL) fixation was up-regulated, while in this study we found and malondialdehyde (MDA), thus reducing membrane that in the drought-tolerant Eruca line DEGs for carbon injury caused by abiotic stresses [67]. Overexpression of fixation in photosynthetic organisms were all down- banana Aquaporin gene MaPIP1;1 increased salt toler- regulated under drought stress (Additional file 5). Ribo- ance in Arabidopsis [68]. In this study one DEG for somes are places where proteins are synthesized. aquaporin TIP1–1 (c194457 g2 i2) was most significantly Dhindsa and Bewley [86] found decline of amino acid in- up-regulated (Additional file 6). corporation and polysome population in the drought- Cuticular waxes play important roles in reducing non- tolerant moss Tortula ruralis under drought stress. In stomatal water loss under stresses [69]. C-5 sterol desa- this study KEGG pathway analysis indicated that DEGs turase catalyzes the incorporation of C-5 double bond involved in ribosome, ribosome biogenesis, Aminoacyl- into D7-sterols to form D5, 7-sterols. Overexpression of tRNA biosynthesis were all down-regulated in drought fungal C-5 sterol desaturase increased wax deposition tolerant Eruca under PEG-simulated drought stress and drought tolerance in tomato [70]. In this study one (Additional file 5); Blast analysis also indicated that 9 of DEG for C-5 sterol desaturase (c201142 g1 i2) was most 10 DEGs for translation initiation factor, 8 DEGs of ATP significantly up-regulated (Additional file 6). synthase, 6 DEGs for ribosomal RNA gene, 5 of 6 DEGs O-acyltransferase WSD1 is also involved in cuticular for eukaryotic translation initiation factor, 118 of 121 wax biosynthesis [71]. Zhang et al. [35] indicated that DEGs for ribosomal protein, were down regulated, KCS and WSD, and their up-stream regulators, were up- among which 38 DEGs for ribosomal protein were most regulated under drought stress. In this study one DEG significantly down-regulated (Additional file 6), suggest- for O-acyltransferase WSD1-like (c175262 g2 i1) was ing that Eruca might also respond to drought stress by most significantly up-regulated (Additional file 6). down-regulation of protein synthesis and carbon fixation in photosynthetic organisms.

Trehalose, galactinol and sugar transport Other pathways and DEGs possibly related to drought In plants various abiotic stresses lead to sugar accumula- tolerance tion in the vacuole [72–74], suggesting that sugar bio- In this study it was found that two DEGs for low- synthesis and vacuolar sugar transporters play important temperature-induced 65 kDa protein-like (c183129 g1 i2, roles under these conditions. Sugar transporter ERD6 c183129 g3 i4), one for lipase ROG1 (c185493 g1 i1), expression is induced by drought stress [75], while one for disease resistance protein (c192485 g1 i11), one ERD6-like transporter (ESL1) expression is enhanced by for amino-acid permease BAT1 (c205647 g1 i3), one for drought and ABA treatment [76]. In this study 2 DEGs ankyrin repeat-containing protein (c201331 g1 i3), one for sugar transporter ERD 6-like 12 (c197650_g1_i3, for acyl-activating enzyme 19 (c188918 g1 i1), one for c197650_g1_i5) were most significantly up-regulated phospholipid hydroperoxide glutathione peroxidase 6 (Additional file 6). (c195141 g2 i3), one for potassium transporter 9-like Huang et al. BMC Plant Biology (2019) 19:419 Page 8 of 11

(c197830 g3 i1), one for phosphoglycerate mutase-like Methods protein 2 (c190346 g1 i4) were most significantly up- Tissue sampling, RNA extraction, library preparation and regulated (Additional file 6). Their roles in drought tol- Illumina sequencing erance need to be further explored. Eruca seeds of PI 426649 and PI 426652 originally from KEGG pathways in which all DEGs were up-regulated, the Agricultural Research Service, USDA were germi- such as Tyrosine metabolism, Phenylalanine, tyrosine and nated on filter paper immersed in liquid MS medium tryptophan biosynthesis, Galactose metabolism, Isoquinoline [87] without sugar or organic components. Seven days alkaloid biosynthesis, Tropane, piperidine and pyridine alkal- later the seedlings were treated for 10 h with 20% PEG- oid biosynthesis, Mineral absorption; KEGG pathways in 6000/liquid MS and then harvested and frozen imedi- which most DEGs were up-regulated, such as Porphyrin ately in liquid nitrogen and then stored at − 80 °C. and chlorophyll metabolism, Ubiquinone and other Drought-tolerant PI 426649 is denoted as ‘DT and terpenoid-quinone biosynthesis, Arachidonic acid metabol- Drought-sensitive PI 426652 is denoted as ‘DS’. Treat- ism, Glutathione metabolism, glycerophospholipid metabol- ment with liquid MS medium is denoted as ‘MS’ while ism, Phenylalanine metabolism, Plant-pathogen interaction, treatment with PEG is denoted as ‘PEG’. Four samples were up-regulated (Additional file 5). Blast analyses indi- (DT-MS, DT-PEG, DS-MS, DS-PEG) each with two bio- cated that all 5 DEGs for dehydration-responsive element- logical replicates were taken for the present study. Total binding protein, 9 of 10 DEGs for glutathione S-transferase, RNA was isolated from the whole seedlings that had 8of10DEGsfortranscriptionfactorMYB,4of5DEGsfor been stored at − 80 °C by using TRIZOL total RNA ex- calcineurin B-like protein, all 16 DEGs for receptor-like traction reagent (TAKARA) according to the manufac- protein kinase, 15 of 17 DEGs for zinc finger CCCH turer’s protocol. RNA integrity was verified by 1.5% domain-containing protein, all 14 DEGs for F-box protein, Agrose gel electrophoresis and confirmed using a 2100 were up-regulated (Additional file 6). These up-regulated Bioanalyzer analyzer (Agilent, CA, USA). The mRNA pathways and DEGs might also play important roles in enrichment, RNA fragmentation, the first and second drought tolerance in Eruca. strand cDNA synthesis and purfying, sequencing adap- tors ligation and PCR amplification were performed as Conclusions described [14]. The libraries were applied to Illumina se- Based on the transcriptomic analyses we postulated that quencing platform (HiSeq 2000, SanDiego, CA, USA) for the most significantly up-regulated DEGs for Ethylene- high-throughput sequencing using a paired-end read responsive transcription factors, WRKY and bHLH tran- protocol with 100 bp of data collected per run. scription factors involved in stress signaling and ABA responses; DEGs for calmodulin-binding transcription De novo sequence assembly, clustering and homology activator, cysteine-rich receptor-like protein kinase, search mitogen-activated protein kinase kinase and WD repeat- After sequencing, the raw image data was transformed containing protein involved in antioxidants and ROS into sequence data by base calling using CASAVA pack- modulation; DEGs for OPDA reductase and allene oxide age provided by Illumina and saved as raw reads of fastq cyclase involved in JA production, C-5 sterol desaturase format and then treated with Trimmomatic(v0.30) to get involved in producing D5, 7-sterols, aquaporin involved in clean reads. Transcriptome de novo assembly was car- decreasing ion leakage and malondialdehyde, O- ried out with short reads assembling program-Trinity acyltransferase WSD1 involved in cuticular wax biosyn- (r2013-02-25) [88]. Unigenes from each sample’s assem- thesis; trehalose-phosphate phosphatase, galactinol synthase bly were taken into further process of sequence splicing 4andsugartransporterERD6-like12involvedinosmopro- and redundancy removing to acquire non-redundant tectant production, up-regulation of DEGs involved in unigenes as long as possible by TGICL [89]. alpha-linolenic acid metabolism, and down-regulation of carbon fixation and protein synthesis might be critical for Functional annotation the drought tolerance in Eruca line PI 426649. These results Blastx alignment (E-value < 0.00001) between unigenes might be valuable for revealing mechanism of drought toler- and protein databases like Nr (NCBI non-redundant ance in Eruca and for improving drought tolerance in crops. database), KEGG (Kyoto Encyclopedia Of Genes and Ge- nomes, www.genome.jp/kegg/) and COG (Clusters of Materials and methods Orthologous Groups of proteins, http://www.ncbi.nlm. Materials nih.gov/COG/) is performed as described [90]. After Nr In this work Eruca vesicaria subsp. sativa PI 426649 annotation, Blast2GO program was used to get GO an- highly tolerant and PI 426652 highly sensitive to PEG- notation for the unigenes. WEGO software was used to simulated drought stress [19] were used for transcrip- do GO functional classification for all unigenes and to tomic analysis. understand the species distribution of gene functions at Huang et al. BMC Plant Biology (2019) 19:419 Page 9 of 11

the macro level [91]. KEGG pathway annotation was car- FPKM: Fragments per Kilo base of transcript per Million mapped reads; ried out by using bidirectional best hit method (BBH) at GO: Gene ontology; GolS: Galactinol synthase; IL: Ion leakage; JA: Jasmonic acid; KAAS: KEGG Automatic Annotation Server; KEGG: Kyoto Encyclopedia Of KEGG Automatic Annotation Server (KAAS). Genes and Genomes; MDA: Malondialdehyde; MS: Murashige-Skoog; NGS: Next generation sequencing; Nr: NCBI non-redundant database; Analysis of differentially expressed genes (DEGs), GO and OPDA: 12-oxophytodienoate; PEG: Polyethylene glycol; q-RT- PCR: Quantitative RT-PCR; ROS: Reactive oxygen species KEGG pathway enrichment Reads of the drought tolerant and sensitive Eruca acces- Acknowledgements sions with PEG/MS treatment were mapped back to our Thanks to GENEWIZ at Suzhou, China, for helping with the transcriptional de novo assembling results using RSEM [92]. To evaluate analysis. the gene expression, the number of unique match reads Authors’ contributions was calculated and then normalized to FPKM (Fragments HB, LZ and ZX conceived the experiments and finalized the paper, HB-L per- per Kilo base of transcript per Million mapped reads) and formed most of the experiments and prepared the draft manuscript, LX, LP, ML and WW helped with material preparation, RNA isolation, qRT-PCR and then used to calculate the unigene expression with re- data analyses. All authors read and approved the final manuscript. strictive conditions of | log2 Ratio | ≥1.0 and FDR ≤ 0.05. GO enrichment analysis of these DEGs was performed Funding using blast2GO with P-value ≤1 and pathway enrichment Material collection and drought tolerance evaluation in this study were financially supported by the National Key Research and Development analysis was carried out using Path finder software against Program of China (2016YFD0100202–23; 2017YFD0101701); transcriptomic the KEGG database with Q-value ≤1. sequencing and data analyses were supported by the National Natural Science Foundation of China (31771829).

Validation of the differentially expressed genes by qRT-PCR Availability of data and materials To confirm the RNA-Seq result some transcript tags The datasets generated and/or analysed in this study are available from the were selected for quantitative RT-PCR (qRT-PCR) ana- corresponding author on reasonable request. lysis. The qRT-PCRs were performed in triplicate ac- Ethics approval and consent to participate cording to the Bioer (Hangzhou, China) manufacturer Not Applicable. instructions and relative gene expression levels were de- ΔΔ termined by the 2- CT method [93]. Primers listed in Consent for publication Additional file 9 for some DEGs were disgned and syn- Not applicable. thesized by GenScript (Nanjing) Co, Ltd. for qRT-PCR. Competing interests The authors declare that they have no competing interests. Additional files Author details 1Hubei Collaborative Innovation Center for Green Transformation of Additional file 1: Table S1 Total GO enrichment. (DOCX 16 kb) Bio-Resources, College of Life Science, Hubei University, Wuhan 430062, 2 Additional file 2: Table S2 KEGG Pathways and Genes Enriched. China. Jiangsu Microbe Biological & Environmental Engineering Co., Ltd, 3 (DOCX 35 kb) Wuxi 214122, China. Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Additional file 3: Figure S1 DEGs in the DS-MS vs DS-PEG group Academy of Agricultural Sciences, Wuhan 430062, China. 4National Key Lab enriched in 14 KEGG pathways. (DOCX 137 kb) of Crop Genetic Improvement, College of Plant Science and Technology, Additional file 4: Figure S2 DEGs in the DT-MS vs DT-PEG group Huazhong Agricultural University, Wuhan 430070, China. enriched in 8 KEGG pathways. (DOCX 111 kb) Additional file 5: Table S3 KEGG pathway enrichment for DEGs Received: 22 February 2018 Accepted: 29 August 2019 specifically in DT-MS vs DT-PEG. (DOCX 19 kb) Additional file 6: Table S4 Differentially expressed unigenes specifically in DT-MS vs DT-PEG based on BLAST analysis. (DOCX 202 kb) References 1. Boyer JS. Plant productivity and environment. Science. 1982;218:443–8. Additional file 7: Figure S3 COG enrichment for DEGs most 2. Mittler R. Abiotic stress, the field environment and stress combination. significantly-up regulated specifically in DT-MS vs DT-PEG group. (DOCX Trends Plant Sci. 2006;11:15–9. 40 kb) 3. Mittler R, Blumwald E. Genetic engineering for modern agriculture: Additional file 8: Figure S4 KEGG pathway enrichment for DEGs most challenges and perspectives. Annu Rev Plant Biol. 2010;61:443–62. significantly-up regulated specifically in DT-MS vs DT-PEG group. (DOCX 4. Price AH, Cairns JE, Horton P, Jones HG, Griffiths H. Linking drought- 58 kb) resistance mechanisms to drought avoidance in upland rice using a QTL Additional file 9: Table S5 Primers for quantitative RT-PCR. (DOCX 16 approach: progress and new opportunities to integrate stomatal and – kb) mesophyll responses. J Exp Bot. 2002;53:989 1004. 5. Shinozaki K, Yamaguchi-Shinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol. Abbreviations 2003;6(5):410–7. ABA: Abscisic acid; AQPs: Aquaporins; BBH: Bidirectional best hit method; 6. Shinozaki K, Yamaguchi-Shinozaki K. Gene networks involved in drought bHLH: Basic helix-loop-helix; CAMTA: Calmodulin-binding transcription stress response and tolerance. J Exp Bot. 2007;58:221–7. activator; COG: Clusters of Orthologous Groups of proteins; CRKs: Cysteine- 7. Hao Z, Li X, Liu X, Xie C, Li M, Zhang D, et al. Meta-analysis of constitutive and rich receptor-like kinases; DEG: Differentially expressed gene; DS: Drought- adaptive QTL for drought tolerance in maize. Euphytica. 2010;174(2):165–77. sensitive; DT: Drought-tolerant; ERD: Early responsive to dehydration; 8. Morozova O, Marra MA. Applications of next-generation sequencing ERF: Ethylene-responsive transcription factors; FDR: False Discovery Rate; technologies in functional genomics. Genomics. 2008;92(5):255–64. Huang et al. BMC Plant Biology (2019) 19:419 Page 10 of 11

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